Bacteria use a sophisticated, two-component, signal transduction mechanism to control specific gene expression that enables adaptation to physical and environmental changes[1]. The system is composed of a histidine kinase and a response regulator. Because (i) signal transduction occurs via a different mechanism in eukaryotes and (ii) some signal transduction events may play a role in virulence[2], histidine kinases are a potential target for new antimicrobial agents[3]. The genome of Burkholderia pseudomallei contains over 20 genes that may be histidine kinases. The predicted periplasmic domain of many of these gene products have been nominated by Dr. Sam Miller (University of Washington) and are approved SSGCID Community Request Targets. The periplasmic domain is the exterior sensory domain that responds to external stimuli (usually ligands but also light, osmotic pressure, pH, and other variables). Due to the wide variety of environmental stimuli that histidine kinases detect, the amino acid sequences of the sensory domain are variable as are the structures reported for some of these domains. In order to use amino acid sequence information to predict stimulus, more structures of the sensory domains of histidine kinases are required along with the identification of the associated stimulus. Such correlation of structure with stimulus will also provide for a better understanding of virulence biochemistry and assist structure-based drug design targeting histidine kinases. SSGCID has determined the crystal structures for the periplasmic domain of one of the targeted B. pseudomallei histidine kinases, BupsA.00863.i (RisS). Indeed, five crystal structures have been solved under different precipitant conditions (3LR0, 3LR3, 3LR5, 3LR4, and 3TE8). A major variable in the precipitant conditions was pH and a significant change in the orientation of the two molecules in the dimer was observed between neutral (6.5) and low (4.5) pH. Such structural changes as a function of pH suggests that part of the biological function of RisS may be to sense low pH, a condition encountered in the acidic environment of macrophage phagosomes. Such a mechanism has been reported for the bacterial sensor kinase PhoQ from Salmonalla typhimurium where PhoQ-mediated gene expression is activated by low pH[4]. Attempts to verify this hypothesis in Dr. Millers laboratory (University of Washington) were conducted by measuring expression levels of RisA, a hypothetical histidine kinase related to RisS, in B. thailandensis E264 cell cultures grown under conditions of different pH, divalent cations, and antimicrobial peptides. Effects were seen only with different levels of divalent cations that showed a 3.5-fold increase in expression in the presence of 10 mM CaCl2. Here, we propose to repeat some of these biological experiments with RisS in Dr. Millers laboratory, but, to assist the guidance of further time-consuming in vivo biochemical studies, we will thoroughly characterize RisS response to various stimuli (pH, divalent cations, and antimicrobial peptides) using isothermal titration calorimetry (ITC) and a suite of NMR-based in vitro experiments.

Work to be performed in the laboratory of Dr. Buchko at WSU and will be carried out during Q1-Q2.

The binding affinity of RisS for a series of microbial peptides and divalent metal ions will be determined using isothermal titration calorimetry (ITC) to identify which, if any, cations and peptides RisS binds to significantly. For the ligands with the best binding affinity, NMR chemical shift perturbation data will be collected. Preliminary data shows that the 1H-15N HSQC spectrum for RisS contains good chemical shift dispersion in both dimensions illustrating that it will be possible to assign the chemical shifts for this protein and obtain residue specific information on the protein from the perturbation study that may be correlated (mapped) with the available crystal structures. Hence, it will be possible to map the interface of the ligand-RisS surface or residues in regions of the protein that occur structural changes. Note that a similar NMR approach was previously used to provide evidence that S. typhimurium PhoQ acts as a pH sensor, however, in comparing 1H-15N spectra at neutral and low pH, the authors only looked for the shifting of cross peaks in the data collected under the two conditions.[4, 5] We propose to go one step further and identify any perturbed resonances by assigning the 1H-15N HSQC spectrum for RisS. Due to the size of the sensor domain, ~ 30 kDa, we observed poor magnetization transfer in most of the 3D backbone NMR assignment experiments collected on a double-labeled sample (13C, 15N). We surmise this is the reason Prost et al. did not make the assignments in their studies. We have solved this problem by preparing a triple-labeled sample (2H, 13C, 15N) that improves the magnetization transfer in the 3D backbone assignment experiments. The preparation of such a deuterated sample, coupled with data collection at elevated temperature (40ºC), will enable us to make the necessary chemical shift assignments and map ligand binding interfaces and/or structural changes onto the available RisS crystal structures.

Work to be performed in the laboratory of Dr. Buchko at WSU and will be carried out during Q2-Q3.

While knowing the structure of a protein is important, proteins are dynamic molecules. Understanding dynamics, especially around active sites, is important for understanding biochemical mechanisms. Upon assigning the 1H-15N HSQC spectrum for RisS it will also be possible to probe the motion of individual protein residues on the picosecond (or larger) timescale using a suite of NMR experiments (T1, T2, and ({1H}-15N-heteronuclear NOEs) [6] and determine the role protein dynamics play in the histidine kinase’s ability to identify specific stimuli.

Summary: Bacteria use a two-component (histidine kinase and a response regulator) signal transduction mechanism to control specific gene expression in repsonse to physical and environmental changes. Analysis of the Burkholderia pseudomallei genome has identified >20 putative histidine kinases, many of which were approved as Community Request Targets. SSGCID obtained five crystal structures (3LR0, 3LR3, 3LR5, 3LR4, and 3TE8) for the periplasmic domain of one histidine kinase, RisS (BupsA.00863.i) under different conditions. A major variable in the precipitant was pH and there was a significant change in the orientation of the two molecules in the dimer between neutral (6.5) and low (4.5). These structural changes suggested that part of the biological function of may be to sense low pH, a condition encountered in the acidic environment of macrophage phagosomes. This FUN project sought to identify the biological signal to which RisS responds. During Year 7, we partially completed two of the three Specific Aims, as detailed below. The structural differences observed for RisS at different pHs in the solid-state have been confirmed in the solution-state using liquid NMR spectroscopy. The solution- and solid-state observations will be assimilated into a single manuscript that provides a structural model on how RisS may act as a pH sensor.

Partially completed. 15N-labelled RisS (BupsA.00863.i) was prepared to optimize conditions for NMR data collection for the amide backbone assignments (see below) and for use in isothermal titration calorimetry and chemical shift perturbation studies. Additional NMR data were also collected to verify that the protein existed as a dimer in solution. Initial ITC experiments to measure disassociation constants suggested weak (in the mM range) binding of divalent cations, so NMR methods were used to measure the Kd. Mg2+ titration studies using 15N-labelled sample (at 37°C) and 2H-, 13C-, and 15N-labelled sample (at 30°C), showed that only two amide resonance shift marginally at a high Mg2+:RisS molar ration (>20:1). A titration with the antimicrobial peptide LL37 was also performed. At the first titration point, a LL37:RisS molar ratio of 0.5:1, visible precipatate appeared. While this indicates the peptide interacts with RisS, it is not known if this is biologically relevant.

Partially completed. In Quarter 2, a 2H-, 13C-, and 15N-labelled sample of RisS was prepared to allow NMR assignment of the “fingerprint” 1H-15N HSQC spectrum of the protein. This special labeling (replacing the protons with deuterium) made it possible to assign the fingerprint 1H-15N HSQC spectrum of the protein at pH 7.0 and 30°C. Only five resonances in the 1H-15N HSQC spectrum could not be assigned, indicating that many of the amide resonances are “invisible”. Indeed, ~33% of the amides are missing or unassigned in the 1H-15N HSQC spectrum. When these resonances are mapped onto the three-dimensional structure of the protein, it is apparent that most of these missing or unassigned resonaces are at the dimer interface. Hence, this dimer interface is either heterogenous or has motion unfavorable for the NMR time-scale. It was not possible to obtain an 1H-15N HSQC spectrum for RisS at low pH because the protein precipitated below pH 5.5. However, it was possible to obtain an 1H-15N HSQC spectrum at pH 5.9. An overlay of the spectra at the two pHs show that a subset of resonances shifts, and more interestingly, four new resonances appeared. Mapping of the resonances that shift at the lower pH onto the structure of RisS showed that most of the perturbations are confined to the regions predicted from the differences in the crystal structures.